Project Summary This is a proposal for an administrative supplement to the parent grant, U01 DK062422, ?IBD Genetics Research Consortium, Genetics Research Center (GRC)?. We propose studies to quantitate protein surface markers for the most impactful pathways in ileal Crohn's disease, while simultaneously measuring transcriptomes in the same cells (CITE-Seq). In previous studies, we have performed single cell RNASeq and defined two types of inflamed tissues, distinguished in part by inflammatory mononuclear phagocytes. Multiple lines of evidence have established that pathophysiologic heterogeneity is substantially defined by innate immune-stromal cross-talk. However, for many of the most impactful Crohn's disease loci, including the IL23R, TNFSF15, and PTGER4 loci, the precise cellular and mechanistic downstream consequences have yet to be completely defined; we hypothesize that this will require more precise distinctions between often closely related immune cells. The premise of this supplemental proposal is that quantitation of GWAS-implicated surface proteins, such as IL23R, TNFRSF25 (TNFSF15 receptor) and PTGER4, none of which are classic lineage markers, will illuminate key cellular differentiation states that underlie GWAS-defined risk alleles' pathogenic roles. Following optimization of cell isolation methods and surface marker selection (Aim 1), we will apply optimized CITE-Seq protocols to Crohn's disease resection tissues. Optimized models integrating clusters best defined by mRNA vs. proteins (e.g. CD4+ vs. CD8+ T cells) will be performed and GWAS- prioritized surface protein expression quantitated by antibody and cellular counts (Aim 2). Finally, imputation of single cell-based insights onto larger datasets will be performed by developing new expression quantitative traits to further define pathophysiologic heterogeneity (Aim 3). Three sources of heterogeneity will be explored. First, we will test for similarities and differences in the IL-23 vs. TNFSF15 pathways; these two pathways demonstrate markedly different variance accounted for between European ancestry and Far East Asian Crohn's disease. Second, we will refine differences between PTGER4 and PTGER2 mRNA and protein- expressing cells. Finally, we will re-analyze European ancestry Crohn's disease case-control GWAS datasets, excluding NOD2 risk alleles, to explore whether the resulting genetic architecture more closely resembles Far East Asian vs. African-American Crohn's disease. By integrating unique insights generated by small (single cell analyses), intermediate (bulk mRNA datasets with matched clinical outcomes) and large (GWAS) datasets, we seek to define the cellular and molecular mechanisms underlying pathophysiologic and clinical outcome heterogeneity in Crohn's disease.